Normal development and functions of immune cells require adenosine deaminase (ADA) activity. The absence or low levels of ADA results in human disease, severe combined immunodeficiency (SCID), which is characterized by hypoplastic thymus, T lymphocyte depletion, and autoimmunity. Accumulation of intracellular adenosine and deoxyadenosine and their direct lymphotoxicity are believed to be responsible for the detrimental effects on T cells observed in ADA SCID patients; however, mechanisms of uniquely high susceptibility of immune cells to effects of ADA deficiency have not been completely understood. We proposed that T cell depletion in ADA SCID patients could be explained by inhibition of TCR signaling in the ADA-deficient environment by extracellular adenosine. Experiments to test this hypothesis revealed that adenosine, but not the deoxyadenosine, inhibits the antigen receptor (TCR)-triggered activation of T cells in vitro . Importantly, the strong inhibition of TCR-triggered activation of T cells of genetically engineered ADA gene-deficient mice was also observed in vivo. This suggests the novel explanation of T cell depletion during the pathogenesis of ADA SCID as due to inhibition by adenosine of T-cell activation critical for processes of thymocytes maturation, emigration from thymus, and T cell effector functions on periphery.. We found that in vivo injection of anti-CD3 mAb strongly activates T cells of ADA-expressing mice but not of their ADA-deficient littermates. In control experiments, the large proportion of ADA-deficient T cells not activated in vivo did respond by activation to TCR crosslinking in vitro, but were inhibited by the addition of extracellular adenosine even in the absence of ADA inhibitor. In addition, results of experiments with ADA-/- T cells confirm that no enzyme in T cells other than ADA degrades extracellularly added adenosine. These studies of ADA+/+ and ADA-/- littermates allowed for previously unavailable controls in investigations of ADA SCID and suggest impairment of TCR-mediated signaling by adenosine in an ADA-deficient environment in vivo as the additional explanation of the unique susceptibility of thymocytes and T cells to the effects of increased concentrations of adenosine. The observations of the "rescue" of adenosine-exposed thymocytes from TCR-triggered apoptosis by extracellular adenosine in ADA deficient environment provided further support of signaling model of ADA SCID vs the intracellular lymphotoxicity of adenosine in these conditions. The finding of A1, A2a and A3 receptor-independent effects of extracellular adenosine in conditions of ADA deficiency suggests the need to investigate the possibility of involvement of A2b receptor and/or other yet to be identified receptor in affecting the activation of ADA deficient lymphocytes. These data point to the important role of effects of extracellular adenosine on the differentiating immune cells as well on mature T lymphocytes and emphasize the further need in understanding of molecular signaling events that follow exposure of lymphocytes to adenosine.